Phosphorylation of Pex11p does not regulate peroxisomal fission in the yeast Hansenula polymorpha.

Thomas AS, Krikken AM, van der Klei IJ, Williams CP - Sci Rep (2015)

Bottom Line:
Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p.Also, no effect on peroxisome inheritance was observed.Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

ABSTRACTPex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein's trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

f1: HpPex11p is phosphorylated on Serine 174.(A) Coomassie stained SDS-PAGE gel showing a band at ~30 kDa, corresponding to Pex11-His6. Numbers indicate molecular weight in kDa. (B) Sequence coverage obtained for Pex11-His6 from MS analysis. Peptides identified in this approach are underlined. Both Serine 174 (open box) and Serine 161 (grey shaded box) are indicated. Black shading depicts the predicted transmembrane domains. (C) nLC-MS/MS analysis of the modified 171-ELASDDDQNPLDKR-184 peptide identified using MS. (D) Sequence of the 171- ELASDDDQNPLDKR-184 peptide, demonstrating that the additional 79.9 Daltons, corresponding to a phosphate group, is present on Serine 174. Indicated are the b and y ions, as well the modified Serine residue (lower case). (E and F) Phos-tagTM SDS-PAGE (E) or SDS-PAGE (F) and western blotting analysis of lysates from pex11Δ cells (pex11) or pex11Δ cells expressing WT or mutant forms of Pex11p. Blots were probed with antibodies raised against Pex11p. Equal amounts of protein were loaded per lane. The modified form of Pex11p visible in WT cells is denoted with an asterisk.

Mentions:
In order to investigate a potential role of Pex11p phosphorylation in H. polymorpha, we purified the protein and analysed it using mass spectrometry (MS). For this, we employed a version of Pex11p complete with C-terminal His6 tag. This version of Pex11p fully complements the pex11Δ deletion strain (Supplementary Figure S1A and B), demonstrating that it is functional. Pex11p-His6 was purified from an organellar pellet (Fig. 1A) and subsequent MS analysis resulted in efficient sequence coverage of Pex11p (Fig. 1B). Furthermore, we identified two peptides that corresponded to the residues 171-ELASDDDQNPLDKR-184. One displayed the predicted molecular mass of approximately 1615 Dalton, whereas the other displayed a molecular mass gain of 79.9 Dalton, which is indicative of the presence of a phosphate group. Peptide sequencing of this peptide (Fig. 1C,D) revealed that the additional 79.9 Dalton was present on the Serine at position 174, strongly suggesting that Pex11p is phosphorylated at this Serine residue.

f1: HpPex11p is phosphorylated on Serine 174.(A) Coomassie stained SDS-PAGE gel showing a band at ~30 kDa, corresponding to Pex11-His6. Numbers indicate molecular weight in kDa. (B) Sequence coverage obtained for Pex11-His6 from MS analysis. Peptides identified in this approach are underlined. Both Serine 174 (open box) and Serine 161 (grey shaded box) are indicated. Black shading depicts the predicted transmembrane domains. (C) nLC-MS/MS analysis of the modified 171-ELASDDDQNPLDKR-184 peptide identified using MS. (D) Sequence of the 171- ELASDDDQNPLDKR-184 peptide, demonstrating that the additional 79.9 Daltons, corresponding to a phosphate group, is present on Serine 174. Indicated are the b and y ions, as well the modified Serine residue (lower case). (E and F) Phos-tagTM SDS-PAGE (E) or SDS-PAGE (F) and western blotting analysis of lysates from pex11Δ cells (pex11) or pex11Δ cells expressing WT or mutant forms of Pex11p. Blots were probed with antibodies raised against Pex11p. Equal amounts of protein were loaded per lane. The modified form of Pex11p visible in WT cells is denoted with an asterisk.

Mentions:
In order to investigate a potential role of Pex11p phosphorylation in H. polymorpha, we purified the protein and analysed it using mass spectrometry (MS). For this, we employed a version of Pex11p complete with C-terminal His6 tag. This version of Pex11p fully complements the pex11Δ deletion strain (Supplementary Figure S1A and B), demonstrating that it is functional. Pex11p-His6 was purified from an organellar pellet (Fig. 1A) and subsequent MS analysis resulted in efficient sequence coverage of Pex11p (Fig. 1B). Furthermore, we identified two peptides that corresponded to the residues 171-ELASDDDQNPLDKR-184. One displayed the predicted molecular mass of approximately 1615 Dalton, whereas the other displayed a molecular mass gain of 79.9 Dalton, which is indicative of the presence of a phosphate group. Peptide sequencing of this peptide (Fig. 1C,D) revealed that the additional 79.9 Dalton was present on the Serine at position 174, strongly suggesting that Pex11p is phosphorylated at this Serine residue.

Bottom Line:
Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p.Also, no effect on peroxisome inheritance was observed.Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

ABSTRACTPex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein's trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.